Abstract/Summary In this proposal we focus on the Pif1 sub-family of DNA helicases and test the hypothesis that one of their functions is to facilitate DNA replication across naturally occurring barriers. We showed that DNA-bound Rap1 is a barrier to the strand-displacement DNA synthesis activity of DNA polymerase ? and that Pif1 displaces Rap1, allowing DNA replication across the barrier. We hypothesize that proteins bound to the DNA, at telomeres and non-telomeric sites, are displaced by Pif1 to facilitate DNA replication, and that this activity is fundamental and conserved among Pif1 homologues. G-quadruplexes and DNA hairpins can also be obstacles to DNA replication. Pif1 is thought to facilitate replication-fork progression by unwinding G-quadruplexes. However, little is known about this activity of Pif1 in the presence of single-stranded DNA binding proteins (SSBs). We hypothesize that the activity of Pif1 is needed to facilitate DNA replication across G- quadruplexes when SSBs cannot effectively melt the DNA secondary structure. Furthermore, we hypothesize that Pif1 stimulates DNA synthesis by DNA polymerase ? across DNA hairpins. We showed that unwinding of dsDNA by a Pif1 monomer is balanced by a Pif1-dependent DNA rewinding activity. We hypothesize that under physiological conditions rewinding is inhibited by concomitant synthesis of DNA by polymerases. Mutations of Pif1 that affect the balance between unwinding and rewinding will allow us to determine the origin of this newly discovered property. To test these hypotheses, we propose three Aims: 1) determine whether protein obstacles that impede DNA replication are displaced by Pif1; 2) determine whether DNA secondary structures are unwound by Pif1 to facilitate DNA replication; 3) determine the mechanism underlying the Pif1-dependent DNA rewinding activity.